Shape-memory-actuated materials for accelerated healing of orthopedic injuries

ABSTRACT

A three component system for repairing critically sized bone defects having a first shape memory polymer (SMP) component formed as a scaffold that fills the defects, a second SMP component formed as a restricting sleeve that stabilizes and supports osseointegration and osteoconduction, and a third SMP component formed as a two-dimensional cell culture substrate for engineering periosteal grafts.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional ApplicationNo. 61/837,226, filed on Jun. 20, 2013.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under D12AP00271 awardedby Defense Advanced Research Projects Agency (DARPA). The government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to bone substitutes and, moreparticularly, to a shape memory polymer system for repairing bonedefects.

2. Description of the Related Art

The nature of modern insurgency warfare combined with improved survivalrates due to advances in body armor has created increasingly complexchallenges for orthopedic reconstruction of extremity injuries inwarfighters. More than 70% of military-related extremity fractures nowinvolve massive bone loss, termed critically sized defects. Currentgrafting options for these defects have important limitations, describedbelow. Additionally, for some combat-related critically sized defectsthere are no reliable treatments, and more than 7% of severe extremityinjuries result in major amputation. There is a need for revolutionaryfunctional materials that significantly improve control of graftintegration (osseointegration) and bone formation (osteogenesis) duringrepair of critically sized defects.

To repair critically sized defects in the extremities of warfighters,live autograft—bone harvested from the same patient—remains the goldstandard for small defects. But autologous bone is not always an option,particularly when multiple-limb trauma occurs within the same patient orwhen the defect is too large. Furthermore, complications include pain,infection, donor site morbidity, and inefficient repair due to limitedosseointegration and remodeling. Allograft—bone harvested from adonor—provides the best current alternative, but often achieves limitedosseointegration with a failure rate of 60% at 10 years. Synthetic bonegraft substitutes have been developed and have generally been calciumphosphate or calcium sulfate space fillers or cements. Like allografts,synthetic bone graft substitutes lack living cells and function only asa scaffold for bone ingrowth (osteoconduction). Moreover, compared toauto- or allograft, synthetic grafts possess inferior mechanicalstrength and fracture resistance, preventing use in large defects thatrequire rapid loadbearing capabilities. Graft approaches are oftenfurther complicated by damage to the periosteum—the tissue that coversthe outer surface of bone and is critical to graft healing andremodeling.

In addition to the challenges listed above, information from majormilitary medical institutions indicates that a subset of defects cannotbe managed well with any existing treatment. Segmental defects—in whicha segment of bone is missing and there is no continuity of bone withinthe fracture—are a particular problem. There is, therefore, an unmetneed for highly effective bone substitutes. Ideally, these substituteswould conform to the defect, rapidly achieve mechanical propertiessimilar to bone, integrate with neighboring bone, and supportosteoconduction and remodeling.

BRIEF SUMMARY OF THE INVENTION

The present invention comprises a system of shape memory polymer (SMP)materials that, together, enable repair of critically sized bone defectsvia three essential and co-dependent steps, namely: (1) filling ofcritically sized defects, (2) stabilization and support ofosseointegration and osteoconduction, and (3) engineering of periostealgrafts that support osteogenesis and remodeling. The first component isan SMP scaffold that can undergo programmed expansion under simulatedphysiological conditions. The scaffold can easily be manipulated by handto fit within a bone defect and, once in situ, can expand to conform tothe shape of the defect for the purpose of quickly integrating with andproviding mechanical properties comparable to native bone whilesupporting osteoconduction and remodeling. For example, the scaffold canuse an end-linked co-network consisting of poly(ethylene oxide) andpoly(epsilon-caprolactone) diene chains linked together into awell-defined covalent network through photoinitiated addition of thevinyl termini with a multifunctional thiol crosslinker.

The second component is an SMP sleeve that can undergo programmed radialcontraction under simulated physiological conditions. Theosteoconductive biomaterial sleeve employs SMP functionality to contractfor the purpose of stabilizing a defect site while concurrentlypromoting healing as a biomimetic periosteum. The sleeve uses athermoplastic polyurethane.

The third component is a two-dimensional cell culture substrate that canundergo programmed expansion or a change in topography formechanobiological engineering of periosteal sheets in vitro. Thetwo-dimensional SMP substrate employs shape-memory expansion orshape-memory actuated change in topography to provide biomimeticbiomechanical stimulation to stem cells during engineering of periostealsheets in vitro. The substrates uses a co-polymer system incorporatingthe monomers tert-butyl acrylate and butyl acrylate, but the technologyis not limited to this particular monomer system.

The present invention thus provides a coordinated system of orthopaedicsmart materials that a military or civilian trauma surgeon ororthopaedic surgeon (the end user) can use to treat critically sizeddefects. This technology will provide surgeons with a new option incases where suitable treatment options have not previously existed,thereby reducing the number of cases that result in amputation. Inaddition, the technology will provide surgeon with an approach thatallows treatment via a single surgery, thereby avoiding the need formultiple revision surgeries, which are often required with currentapproaches. Furthermore, the resorbable and biodegradable design of thetechnology avoids complications, such as corrosion and local andsystemic infection and cytotoxicity, commonly associated with metalhardware used in many current treatment options. Further applications ofthe technology include adaptation of the system to treatment of injuriesand diseases in both other areas of the musculoskeletal systems and inother organ systems. In addition, the system can be scaled for use byveterinarians in animals.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

The present invention will be more fully understood and appreciated byreading the following Detailed Description in conjunction with theaccompanying drawings, in which:

FIG. 1 is a schematic of a three component system, including a Scaffoldthat can undergo programmed expansion to conform to the shape of a bonedefect and support osteoconduction and remodeling, an osteoconductivesleeve that can undergo programmed radial contraction to stabilize adefect site and promoting healing, and two-dimensional cell culturesubstrate that can undergo programmed expansion or topographic change toprovide biomimetic biomechanical stimulation to stem cells duringengineering of periosteal sheets for use as periosteal grafts;

FIG. 2 is a schematic of a scaffold according to the present inventionthat can undergo programmed expansion to conform to defect shape and tosupport osteoconduction and remodeling;

FIG. 3 is a series of images of a SMP foam prepared according to thepresent invention from end-linked poly(ε-caprolactone) using saltleaching, where the disk is 25 mm in diameter and the SEM scale bar is500 μm;

FIG. 4 is a schematic of a osteoconductive sleeve according to thepresent invention that can undergo programmed radial contraction tostabilize a defect site and promoting healing;

FIG. 5 is a series of images of a nanofibrous sleeve contracting duringheating, where the scale bar is 10 μm is the first image and the scalebar is 500 μm in the remaining images;

FIG. 6 is a schematic of a two-dimensional cell culture substrateaccording to the present invention that can undergo programmed expansionto provide biomimetic biomechanical stimulation to stem cells duringengineering of periosteal sheets for use as periosteal grafts;

FIG. 7 is an image of a sheet of mouse C3H/10T1/2 progenitor cells beingcultured under a gradient of tension on an expanding SMP substrate, andare shown after 13 h of substrate expansion following triggering byslight heating from 30 to 37° C., with a max strain if 8.1%, theapproximate direction of expansion indicated by the double-headed arrow,and phalloidin (green) staining of filamentous actin and SYTOX (blue)staining of nuclei, where the scale bar is 100 μm;

FIG. 8 is a series of scanning electron micrographs of the top surfaceof a PCL scaffold (a) before fixing, (b) after fixing, and (c) afterrecovery;

FIG. 9 is a series of images of a LIVE/DEAD assay performed on cellsseeded on a scaffold with a pore size of 200-500 μm, with (left) cellsin a single focal plane and a (right) z-stack projection of 120 μmshowing cells from multiple focal planes (scale bar 200 μm), where greencells stain viable cells, while red cells depict dead cells;

FIG. 10 is a schematic of SMP network formation using photo-initiatedaddition reactions between oligomeric macromers and the showntetrathiol;

FIG. 11 is a series of images showing compressive shape memory behaviorand effect on pore morphology, with compressive shape memory (left)before compression, (middle) after compression and fixing, and (right)after recovery show that the SMP scaffold is able to fully recover backto the original state and restore its porous architecture, where scalebars: 1 mm (middle row) and 100 μm (bottom row);

FIG. 12 is a graph of the thermogravimetric analysis of 92tBA-8BA foamsaccording to the present invention;

FIGS. 13A and 13B are graphs of the storage modulus sweep for 92tBA-8BAfoams and 80PCL-20PEG foams, respectively;

FIG. 14 is a schematic of the formation of a PDLLA diacrylate; and

FIG. 15 is a schematic of the cross-linking of the functionalized PDLLAdiacrylate of FIG. 14 with tetrathiol via UV polymerization;

FIG. 16 is a graph of the thermogravimetric analysis of PDLLA_POSS_34k;and

FIG. 17 is a graph of the second heating trace and first cooling traceof PDLLA_POSS_34k.

DETAILED DESCRIPTION OF THE INVENTION

Referring now to the drawings, wherein like reference numerals refer tolike parts throughout, there is seen in FIG. 1 a three component systemof shape memory polymer (SMP) materials according to the presentinvention that, together, enable repair of critically sized bone defectsby filling critically sized defects, providing stabilization and supportof osseointegration and osteoconduction, and allowing engineering ofperiosteal grafts that support osteogenesis and remodeling. However, itis also possible for the three components of the present invention to beused individually, or in sub-combinations, depending on the injury andextent of bone loss.

The first component of the present invention is an SMP scaffold 1 thatcan undergo programmed expansion under simulated physiologicalconditions. Referring to FIG. 2, the scaffold can easily be manipulatedby hand to fit within a bone defect and, once in situ, can expand toconform to the shape of the defect for the purpose of quicklyintegrating with and providing mechanical properties comparable tonative bone while supporting osteoconduction and remodeling. Preferably,the first component according to the present invention usestemperature-, hydration-, or degradation-actuated changes in shape todevelop a porous foam scaffold that can be programmed to expand underphysiological conditions. The rationale for this approach is thatexpansion will allow the scaffold to conform to a defect and will resultin high porosity, facilitating osteoconduction. Use of a semi-rigidscaffold, rather than a hydrogel as in conventional approaches, willenable rapid achievement of functional mechanical properties, allowingrapid resumption of loadbearing and speeding healing and remodeling. Ina preferred embodiment, this aspect of the present invention is formedfrom a single polymer family: an end-linked co-networks consisting ofpoly(ethylene oxide) and poly(ε-caprolactone) diene chains linkedtogether into a well-defined covalent network through photo-initiatedaddition of the vinyl termini with a multifunctional thiol crosslinker.This polymer family may be tailored for application as a bone graftsubstitute with structural actuation by slight heating, waterplasticization, polymer degradation, or some combination of the three.Furthermore, the end-linking chemistry can be modified to includeurethane bond formation by functionalizing the macromer chains describedabove with either isocyanate or alcohol groups and crosslinking with amultifunctional, complementary crosslinker bearing alcohol or isocyanategroups, respectively, and using a suitable catalyst from among manyknown in the art. Similarly, the end-linking chemistry can be modifiedto include alkyne-azide “click” chemistry by functionalizing themacromer chains described above with either alkyne or azide groups andcrosslinking with a multifunctional, complementary crosslinker bearingazide or alkyne groups, respectively, and using a suitable catalystknown in the art. Standard methods of polymer science (e.g., x-ray,calorimetry, and microtensile testing) may be applied to ascertainstructure-property relations that guide refined materials design. Porousfoams may be formed by salt leaching, such as end-linked PCL networkswith 10% polymer density, as seen in FIG. 3.

To modulate cell adhesion on such scaffolds, an RGD-bearing networkchain was employed, which has proven effective in companion work onshape memory hydrogels. In addition, nano-hydroxyapatite (HA)incorporation into the scaffold may increase cell adhesion andmineralization. Scaffold storage modulus increased with HA content whilethe degree of shape-memory recovery decreased, as would be expected.Conformation of the scaffold to a defect may be simulated using circularor irregular holes punched in high-HA-content PCL films, with assessmentof anchoring using push-out tests. Minimally invasive delivery may besimulated by passing scaffolds through a 10 mm ID laparoscopic tube into37° C. culture medium, followed by analysis of recovery. Osteoconductivecapacity may be assessed by histology and quantitative polymerase chainreaction following culture on the scaffolds of adipose-derived stemcells (ASCs) in osteogenic medium. As discussed below, other embodimentsusing different SMP compositions are also possible.

The second component is an SMP sleeve 2 that can undergo programmedradial contraction under simulated physiological conditions. Referringto FIG. 4, the osteoconductive biomaterial sleeve employs SMPfunctionality to contract for the purpose of confirming to andstabilizing a defect site while concurrently promoting healing as abiomimetic periosteum and anchoring the sleeve to the intact neighboringbone, thereby simplifying surgical management of the injury. Use of ananofibrous mesh can maximize osteoconduction, graft remodeling, andregeneration of a functional periosteum in situ. By providing acompressive barrier, the sleeve can also contain and stabilizeautograft, allograft, or synthetic bone graft, including the firstcomponent described above. In a preferred embodiment, the secondcomponent comprises a thermoplastic polyurethane (TPU) with abiodegradable and glassy soft segment adapted to obtain an actuationtemperature that can be tailored by adjusting the deformationtemperature, slow degradation over weeks, and cell viability comparableto that of the untreated control. Alternatively, high molecular weightversions of the polyol (soft segment) itself—poly(D,L-lactide) may beused. Here, the molecular weight must be sufficiently high to allow forboth electrospinning of nanofibers and also entanglement-basedelasticity (achieved in the TPU by hard-block crystallization) requiredfor shape contractions upon stimulation. As another alternative,poly(vinyl acetate) may be used. Nanofibrous sleeves may be prepared byelectrospinning and engineered to contract in response to stimulation.The changes in shape of sleeves may be quantitatively characterized whenfixed in a radially expanded state and actuated to contract by slightheating, hydration, and/or degradation, such as those seen in FIG. 5.Contraction and stabilization of a defect may be simulated and assessedby imaging recovery of scaffolds around a mock defect, for which twodowels can be used, with assessment of anchoring by pull-out tests.Osteoconductive capacity may be assessed as described above.

The third component is a two-dimensional cell culture substrate 3 thatcan undergo programmed expansion or topographic change formechanobiological engineering of periosteal sheets in vitro. Thetwo-dimensional SMP substrate employs shape-memory expansion to providebiomimetic biomechanical stimulation to stem cells during engineering ofperiosteal sheets in vitro. The substrates use a co-polymer systemincorporating the monomers tert-butyl acrylate and butyl acrylate.Alternatively, the commercially available optical adhesive, NOA-63, andsimilar formulations may be used for this purpose, the sole requirementbeing that the polymer system be glassy, crosslinked, and feature aglass transition temperature tunable in the 30-50° C. range. As seen inFIG. 6, substrate expansion will provide attached stem cells withtensile stimulation that mimics that present during in vivo development.Control over the actuation temperature can be achieved by systematicallyvarying the weight percentage of tBA and BA in the copolymer whilekeeping the crosslinking density constant. It is possible toquantitatively characterize gradient substrates that expandnon-uniformly (when fixed following cantilever bending) when actuated byslight heating, as seen in FIG. 7. To determine optimal expansionmagnitude and rate, ostogenic induction may be assessed on high-densitycell sheets cultured on the gradient samples, using the assays describedabove. Both human ASCs and the multipotent mouse embryonic fibroblastscell line C3H/10T1/2 may be assessed. Cell sheets will be harvestedfollowing culture using from the substratum using a cell scraper. Cellsheets will then be cultured on cell culture dishes and on flat samplesof the sleeve according to the present invention and characterized forosteogenic and osteoconductive capacity, as described above.

As explained herein, various compositions that can serve as eachcomponent were prepared and tested to determine shape memorycharacteristics as well as to glean the particular cellular interactionsnecessary for the use of the present invention.

Example 1—SMP Scaffold

Foam scaffolds were fabricated using a modified porogen-leachingtechnique. A functionalized poly(ε-caprolactone) (PCL) macromer and asecond functionalized hydrophilic macromer based on poly(ethyleneglycol) (PEG) were mixed with salt in a 9:1 salt-to-polymer ratio andcrosslinked via thiol-ene chemistry using a photoinitiator. Aftersynthesis the salt particles were extracted using water, creating aporous foam scaffold with high porosity.

The ability of the foam scaffold to fill space was determined bycharacterizing its shape memory behavior using a dynamic mechanicalanalyzer (DMA). Circular discs were heated above their meltingtransition temperature, compressed, cooled to fix the temporarydeformation, and then heated back above the melting temperature toexpand to the original shape. The resulting fixing and recovery ratioswere measured.

The ability to tune the recovery temperature to a physiologicaltemperature was investigated via two methods. First, the ratio of thetwo macromers during synthesis was systematically varied and theresulting melt transition was determined using a differential scanningcalorimeter (DSC) of the scaffolds in the dry and hydrated states.Second, the temperature at which the scaffolds were deformed during theshape memory process was varied and the resulting onset of recovery wasmeasured using a DMA.

Pore architecture and interconnectivity was investigated usingmicrotomography (μCT) and the shape memory effect on the porousstructure was analyzed using scanning electron microscopy (SEM).Preliminary cell culture studies were performed to test thecytocompatibility of the scaffolds. Human adipose derived stem cells(hASCs) were seeded on the material and assayed using SEM and Live/Deadstaining 2 d and 4 d after seeding.

Thermomechanical testing showed that the foam had excellent compressiveshape memory properties: the shape fixing and recovery ratios of thematerial were 99 and 95%, respectively, as determined from the one-wayshape memory cycle (data not shown). The foam also revealed a highcompression ratio as it was able to be compressed to ˜25% of itsoriginal thickness without failure and with recoverability.

Tuning of the transition temperature was achieved by varying the weightratio of the two functionalized macromers during fabrication and varyingthe deformation temperature during the shape memory cycle. It wasobserved that, for samples hydrated to equilibrium, as the weightfraction of the hydrophilic macromer was increased the resulting meltingtransition temperature as measured from DSC decreased, with a range nearbody temperature. Also, as the deformation temperature decreased, theresulting onset of recovery decreased. However, decreasing thedeformation temperature also led to a decrease in fixing ratio (data notshown).

Pore microstructure was observed using uCT analysis and it was foundthat scaffolds with porosities >80% and high interconnectivities wereachieved (data not shown). SEM analysis of scaffolds prior to fixing,after fixing, and after recovery revealed that macroscopic compressionled to microscopic compression of the pore structure. Gratifyingly, thismicrostructural transformation was subsequently restored upon recovery.

Cell culture experiments were conducted to test cytotoxicity of thescaffolds. SEM analysis after 2 d and 4 d revealed cells attached andwere well spread in the scaffold (data not shown). Live/Dead analysisrevealed cells remained viable after 2 d and 4 d and cells hadproliferated on the scaffold (data not shown).

This embodiment of the present invention thus provides a cytocompatiblescaffold capable of being fixed in a compressed state and recovering toexpand to the original state at a physiological temperature. Thescaffold showed excellent shape fixing and recovery in compression andthe transition temperature was able to be tuned through compositionvariation and control of the deformation temperature.

Example 2—SMP Scaffold

Another embodiment of the present invention involves a crosslinkedpoly(caprolactone) (PCL) scaffold prepared using a porogen-leachingtechnique. A functionalized PCL macromer was mixed with salt in a 9:1salt-to-PCL ratio and crosslinked with tetrathiol. After synthesis thesalt particles were extracted using water, creating a porous foamscaffold with high porosity.

Following salt extraction, the melting temperature of the scaffold wasdetermined using differential scanning calorimetry (DSC), and the shapememory properties characterized using dynamic mechanical analysis (DMA).Samples were heated above their melting temperature, uniaxiallystretched, cooled to fix the temporary deformation, and then heatedabove their melting temperature to recover to the permanent shape. Theresulting fixing ratio (how much of the deformed strain is maintainedupon fixing) and recovery ratio (how much of the temporary strain can berecovered) were measured. Pore morphology was investigated usingscanning electron microscopy (SEM) at each stage of the shape memorycycle.

Preliminary cell culture studies were performed to test materialbiocompatibility and attachment. For this study, C3H10T1/2 mousefibroblasts were seeded on the scaffold material. A Live/Dead assay wasperformed to assess cell viability and penetration into the scaffold.

Thermomechanical testing showed that the foam had excellent shape memoryproperties: the shape fixing and recovery ratios of the material were 99and 93%, respectively, as demonstrated by a one-way shape memory cyclefor the PCL foam in tension. With high fixing and recovery percentages,this material is a good candidate for being manipulated to fillcritical-sized defects, though requiring lowering of the transitiontemperature by composition alteration.

To determine pore microstructure, SEM was conducted on the top surfaceof the foam prior to fixing, after fixing, and after recovery, as seenin FIG. 8. As synthesized, the pore structure is round and open. Afteruniaxially stretching and fixing the pores become elongated. Afterrecovery the pores return back to a more rounded morphology.

Cell culture experiments were conducted to test cell viability andpenetration into the scaffold. More than 99% of the cells were viableand cells were seen at depths up to 120 μm, as shown in FIG. 9. Herecells were seeded on a static scaffold that did not undergo any activeshape change. Given the excellent viability and penetration of cellsinto the scaffold as well as the excellent shape fixing and recovery ofthe scaffold, shape recovery is anticipated to present cells withsubstantial biomechanical stimuli. It may be possible to tune suchstimuli to control cell behavior.

This embodiment of the present invention provides a biocompatiblescaffold with shape-changing capabilities. The SMP employed exhibitedexcellent shape fixing and recovery as well as control over poremorphology through the shape memory effect. Cells readily attached,penetrated and remained viable in the scaffold.

Example 3—SMP Scaffold

To prepare highly porous scaffolds using a robust and simple technique,a modified porogen-leaching process was employed, similar to a methodestablished by Zhang and colleagues for PCL-block-PolydimethylsiloxaneSMP foams. Functionalized PCL and PEG macromers were dissolved indichloromethane (DCM) and combined with tetrathiol crosslinker and2,2-dimethoxy-2-phenylacetophenone photoinitiator as seen in FIG. 10.This solution was added to fused NaCl crystals in a 1:9 polymer:saltratio by weight and UV cured. The fusion of salt particles, prior to theaddition of the macromer solution, was performed to improve the poreinterconnectivity. Following curing, salt was extracted in water,yielding highly interconnected porous foams with porosities of 79±5% asdetermined by microtomography. Volumetric shrinkage of scaffolds wasobserved following the first heating cycle and this shrinkage wasdependent on the macromer concentration in DCM.

The shape memory behavior of the scaffold was quantitativelycharacterized using a one-way shape memory compression test. Prior totesting, samples were thermally treated by heating to 80° C. for 10 minfollowed by cooling to −4° C. for 10 min to remove residual stressesgenerated during curing. To test shape memory behavior, circular disksof the scaffold were heated to 80° C. and uniaxially compressed. Whilemaintaining the compressive deformation, the samples were cooled to 0°C. to induce crystallization, immobilizing the chains and fixing thedeformation. Upon unloading, a fixing ratio—how much of the programmeddeformation is maintained upon unloading—of 99±0.5% was observed.Samples were then heated to 80° C. to trigger recovery of the scaffold,with a recovery ratio—how much of the programmed deformation isrecovered upon heating—of 97%±1.4% observed. The programmed stateremained stable at room temperature with no observable prematurerecovery during 6 d of storage. Stability of temporary shapes is adesirable characteristic both for tissue engineering scaffolds and foractive cell mechanobiology studies, in which cell seeding is performedin the temporary state.

To determine the effect of shape memory on macroscopic and microscopicscaffold architecture, scanning electron microscopy (SEM) was performed.Prior to programming the temporary shape, SEM of the scaffoldcross-section revealed an open pore structure with highinterconnectivity. Upon programming the compressive deformation, theporous architecture collapsed as struts began layering on top of oneanother, significantly reducing the porosity. Importantly, the porousarchitecture was restored upon recovery of the scaffold, with no obviouscompromise of the integrity of the internal foam walls, as observedvisually from SEM or mechanically from repeated shape memory cycleswhere the material's modulus did not drop after three repeated cycles.Large compression ratios of up to 78% were achieved, which is desirablefor tissue engineering strategies, such as minimally invasive delivery,or mechanobiological study of large tensile strains on cells seeded infixed scaffolds. For example, in adult cardiac fibroblasts it has beenshown that a 10% uniaxial tensile strain can stimulate extracellularmatrix mRNA levels and transforming growth factor-β (TGF-β), whereas a20% strain decreases extracellular matrix mRNA expression whilestimulating TGF-β to a lesser extent. Ultralow porosity in the fixedstate may inhibit cell infiltration into the scaffold when seeding inthe temporary state. As a result, tissue engineering applications orcell mechanobiology studies for which large strain triggering is desiredwould be expected to have to balance the desired level of strain withthe ability to achieve cell seeding in the fixed, low porosity state.Therefore, to enable studies on increasing levels of strain recovery,increasing porosity in the permanent state will be required. SMPscaffolds fabricated via the alternative method of gas foaming have beendeveloped with permanent porosities of 98%, but with gas foaming poreinterconnectivity is typically low, which would limit cell infiltrationinto such scaffolds. For the salt leaching approach employed in thepresent work, pore size, porosity and interconnectivity can be tunedthrough controlling the size of the salt particles, the degree of saltfusion, and the concentration of macromer in solvent. Control over theporous structure is important for cell mechanobiology studies, asprevious studies on static scaffolds have shown that cell behavior isdependent on pore morphology and size.

Shape-changing scaffolds that can change shape under cell compatibleconditions, particularly at or near body temperature, require controlover the triggering mechanism. Shape recovery of semi-crystalline SMPsoccurs at their melting transition temperature (T_(m)), and the T_(m) ofPCL is ˜60° C., which is much higher than body temperature. Therefore,lowering of T_(m) is necessary to fabricate a shape-changing PCL-basedconstruct. Here, lowering of Tm was achieved via two mechanisms. Thefirst mechanism utilized copolymerization of macromomers of PCL withPEG, a hydrophilic polymer. A PCL-PEG hydrogel with a T_(m) of 31° C.,where tuning of the Tm was achieved through control over the molecularweight of the PCL18, has been reported. Here, the molecular weight ofthe macromers were kept constant and the weight ratios of each varied tocontrol the T_(m).

Differential scanning calorimetry revealed that the T_(m) of thescaffold in both the dry and wet states decreased with increasing PEGcontent. As a consequence of increasing PEG, the water uptake of foamsalso increased. Employing this design strategy, a range of T_(m)'saround body temperature was achieved, with a composition of 80 wt-% PCLand 20 wt-% PEG yielding a hydrated T_(m) of 37° C.

In addition to copolymerizing the PCL scaffolds with PEG for meltingpoint modulation, the programming temperature of the SMP foam was alsovaried to lower the apparent T_(m), or onset temperature for shaperecovery. Scaffolds were heated to different temperatures ranging from30° C. to 80° C. then uniaxially compressed. After reaching either apredetermined strain of 30% or the force limit of the tensile testingdevice, the scaffolds were next cooled to 0° C. to fix the deformationby crystallization. It was observed that for deformations well aboveT_(m) of the construct, there was little dependence of onset temperaturefor recovery on deformation temperature. However, as the deformationtemperature approached T_(m) from above, an associated decrease in theonset temperature was observed. Such a “temperature memory” phenomenonwas previously reported where a large alpha transition related to theionic cluster phase is largely responsible for this effect. A relatedphenomenon has been exploited in amorphous systems to tune the recoverytemperature and recovery kinetics, where deforming at or below the glasstransition temperature led to lower recovery temperatures. Thisphenomenon has also been observed in semi-crystalline polymers whererecovery temperatures spanning a range of 100° C. were achieved. Thepresent invention is the first instance of semi-crystalline SMP foamsexhibiting such temperature memory.

Although deforming near T_(m) results in a lowering of the onsettemperature for strain recovery to within a physiological range, anunintended consequence of this approach is a reduction in the stabilityof the temporary shape at room temperature, examined by dwelling at thattemperature during the heating step of the shape memory cycle, alongwith a reduction in fixing ratio. As the deformation temperaturedecreased, a corresponding decrease in stability was observed. Thereduction in fixed strain stability at room temperature may beattributed to relaxation of internal stresses between the high meltingfraction of the material, which is elastically deformed at the lowertemperature and thus under compressive stress, and the lower meltingfraction, which is otherwise well fixed put into tensile stress by thehigh-melting fraction. Despite these complexities, the temperaturememory effect offers a useful tool to control the recovery temperatureof this SMP foam.

Surprisingly, the foams according to the present invention alsoexhibited two-way reversible shape memory under the bias of acompressive load, consisting of dramatic cooling-induced compression andheating-induced expansion. By inspection, neither effect is due toordinary thermal expansion effects; rather, crystallization of the foamsunder a compressive load results in additional contraction that isreversed upon heating through T_(m), with thermal hysteresis of ca. 50°C. for the heating rate used. This effect is repeatable through severalcool-heat cycles and represents a new example of reversible, softactuation. For these samples, a compressive actuation strain of ca. 15%was achieved through contraction upon crystallization. Interesting, thisactuation strain is non-monotonic in the initial strain applied,indicating competing effects of strain that drivescrystallization-induced actuation, and an upper bound of compressivestrain for the foams. This is the first instance of an SMP scaffold withtwo-way reversible shape memory in compression. Two-way reversibleactuation has been studied in other material systems, such as shapememory alloys, for application in reversible actuators. Incorporatingthis functionality in a cytocompatible scaffold creates the possibilityfor generating cyclic loading on attached cells by simply switching theincubation temperature.

Example 4—SMP Scaffold

SMP scaffolds were fabricated using a modified porogen leachingtechnique, in which a functionalized poly(ε-caprolactone) (PCL) macromerwas mixed with NaCl in a 9:1 salt-to-PCL ratio by weight andsubsequently crosslinked via thiol-ene chemistry. Salt particles werefused for 24 h in a humidity chamber prior to adding the macromersolution. Once the polymer was cured, salt particles were extracted withwater, yielding a porous foam scaffold with high porosity andinterconnectivity. Shape memory characterization of the scaffold wasperformed using a dynamic mechanical analyzer measuring compressivestrain fixing and recovery. The resulting porous architecture beforefixing, after fixing, and after recovery was investigated with scanningelectron microscopy (SEM) and microtomography. Tuning of the functionalrecovery temperature to a cytocompatible temperature was achievedthrough both composition and deformation temperature adjustments. Cellstudies were performed using human adipose derived stem cells toinvestigate cell viability and cell proliferation on the scaffolds.

The porogen-leaching technique yielded polymeric scaffolds featuringporosities >80% with high interconnectivity. These scaffolds exhibitedexcellent shape fixing and shape recovery characteristics, with fixingand recovery ratios of 99% and 95%, respectively. Prior to deformationand fixing, pores were open and interconnected, whereas aftercompressive deformation pore architecture collapsed; pore structure wasable to then recover to the original size and shape after recovery, asseen in FIG. 11. The functional recovery of the scaffolds was easilytuned by adding a second functionalized macromer to the system. Thishydrophilic macromer, once hydrated, served to decrease the recoverytemperature to 37° C. Deformation temperature also served as a viablemethod for controlling the recovery temperature, with lower deformationtemperatures leading to lower recovery temperatures. Preliminary cellstudies using Live/Dead viability assay revealed cells attached andremained viable after 4 d on the scaffold (data not shown). SEM imagingof cells seeded on the scaffold revealed cells proliferated to cover thesurface of the scaffold and cells appeared well spread on the scaffold(data not shown).

Example 5—SMP Scaffold

An SMP scaffold was formed using the aforementioned modified porogenleaching technique, in which tert-butyl acrylate (tBA), butyl acrylate(BA), tetraethylene dimethacrylate (TEGDMA) as a crosslinker, and2,2-Dimethoxy-2-phenylacetophenone (DMPA) as a photoinitiator were mixedin a template of fused NaCl and crosslinked via UV-initiated freeradical polymerization. To fabricate a foam with a hydrated Tg of 37°C., a solution of 92 wt-% tBA and 8 wt-% BA was mixed with TEGDMA (5wt-% relative to the amount of tBA and BA) and DMPA (1 wt-% relative tothe amount of tBA and BA) and added to the salt template. The solutionwas then cured in a UV box for 2 h followed by drying in a vacuum ovenfor 24 h. After drying, salt particles are extracted in 50° C.(temperature above Tg) water for 48 h. As seen in FIG. 12,thermogravimetric analysis of 92tBA-8BA foams reveals than when heatingto 600° C. no salt remains, as the final weight of 6% is the same as forthe films of the same compositions. Samples were heated at 10° C./min to600° C. The Tg-based foam has a stiffness an order of magnitude higherthan the semi-crystalline based system. As seen in FIGS. 13A and 13B,the storage modulus sweep for 92tBA-8BA foams (FIG. 13A) show a storagemodulus an order of magnitude higher than the 80PCL-20PEG foams (FIG.13B) when dry. Note that due to the large stiffness of the tBA/BA foam,the storage modulus drop could not be measured in the DMA with the givenparameters (data ends right as modulus drop begins at 60° C.).

Example 6—SMP Scaffold

In another embodiment of an SMP scaffold seen in FIGS. 14 and 15, aPDLLA diol may be end-capped with an acrylate endgroup, following aprotocol established by Y. Zhang, C. Y. Won and C. C. Chu, Journal ofPolymer Science Part A: Polymer Chemistry, 1999, 37, 4554-4569, herebyincorporated by reference in its entirety. Briefly, a PDLLA diol may bedissolved in anhydrous THF in an ice-chilled flask under nitrogen.Triethylamine in a molar ratio of 4:1 (triethylamine:PDLLA diol) may beadded to the reaction flask, followed by an equimolar amount of acryloylchloride (relative to triethylamine). The reaction may be carried outfor 3 h at 0° C. followed by 18 h at room temperature. This is expectedto yield the highest degree of end-capping according to Zhang.Endcapping of a PDLLA diol having a MW of 10.4 kDa with acryloylchloride following this procedure resulted in incompletefunctionalization and, as a result, the foam was not fully cross-linked.Moisture may have inhibited the reaction as this system is moisturesensitive so formation of this embodiment may require a high degree ofmoisture control.

Example 7—SMP Scaffold

In this embodiment, the scaffold comprises a polyurethane-based shapememory foam where PDLLA is be used as the soft block and POSS is used asthe hard block. The chemistry is the same as that employed for thecontracting sleeve below. To achieve a foam, TPUs with differingmolecular weights may be dissolved in chloroform and vacuum infiltratedinto a fused salt template. After infiltrating into the template, thechloroform may be removed from the system in a vacuum oven. To fix inthe permanent shape of the foam, samples may be heated to 130° C. for 10min to melt the POSS domains. The foams may then be cooled to roomtemperature to allow recrystallization of POSS, setting the permanentshape and removing any residual stress in the material.

Following thermal removal, salt may be extracted from the foams for 24 hin water at 50° C. (a temperature above Tg of the TPU). Foams may thenbe dried and the thermal, thermomechanical, shape memory, anddegradation properties will be characterized. A foam fabricated using aTPU with a molecular weight of 34 kDa was made by first dissolving theTPU in chloroform in a 50% (w/v) concentration, and the vacuuminfiltrating into a template. Salt was completely extracted as shown inthe TGA trace. As seen in FIGS. 16 and 17, thermal characterizationrevealed a dry Tg of 48.9° C., which is expected to decrease ˜10° C.upon hydration, as observed in studies on the contracting sleeve. Foamswith Mn=˜60 kDa and ˜160 kDa may be formulated to investigate the effectof molecular weight on thermal, thermo-mechanical, shape memory, anddegradation behavior.

Example 8—SMP Sleeve

Electrospun scaffold meshes were prepared from a custom synthesizedshape-memory thermoplastic polyurethane and employed as programmablescaffolds as follows: a dynamic mechanical analyzer was used touniaxially stretch scaffolds to 100% strain at 65° C. (above Tg, glasstransition temperature) and fixed at 0° C. in an elongated state. Humanadipose-derived stem cells (ASCs) were cultured on either thetemporarily aligned scaffolds or the previously recovered scaffolds (asa random control) at 30° C. for 24 h. Shape change was triggered byincreasing the temperature to 37° C. Cells were cultured at 37° C. foran additional 24 h.

Scaffold architecture and cell orientation and morphology were assayedbefore and after shape transition by scanning electron microscopy (SEM)and fluorescence imaging. Two-dimensional fast Fourier transform (2DFFT) analysis was used to characterize the alignment of actin filamentswith the corresponding scaffold structure change. The degree of actinfilament alignment was determined by the amplitude and shape of thepeaks in each FFT plot. The higher the peak, the more precisely theactin filaments were aligned along a principle direction.

The programmable shape-changing electrospun scaffold retained an alignedstructure when held at 30° C. (below Tg). The scaffold underwentsubstantial fiber reorientation and pore size alteration during theheat-triggered shape change. The structure change of the programmableshape-changing scaffold directed cell morphology and orientationcompared to the static random control. For cells cultured on atemporarily aligned fibrous scaffold, there are two distinct peaks, at90° and 270°, in the FFT plot compared to cells cultured on thepreviously recovered random fibrous scaffold. After activating shapechange, cells lost their preferential alignment corresponding to fibrousstructure change; no prominent peak was generated in the FFT plot.Fluorescent imaging further showed cytoskeletal filaments reorganizationafter the temporarily aligned fibrous structure recovered back to itspermanent random structure. Cells remained randomly orientated on thepreviously recovered fibrous scaffold.

Example 9—SMP Sleeve

Electrospun scaffolds for use as the second component of the presentinvention were fabricated from a custom-synthesized shape-memorythermoplastic polyurethane and employed as shape-changing scaffolds asfollows. An electrospun scaffold with randomly oriented fiberarchitecture was uniaxially stretched in a dynamic mechanical analyzerto 100% strain at 60° C. (above Tg, glass transition temperature) andfixed at 0° C. in the temporarily elongated shape, which exhibited astrain-aligned fiber architecture. Human adipose-derived stem cells(hASCs) were seeded on the strain-aligned (active) scaffolds as well ason unaligned and aligned control (static) scaffolds, and cultured at 30°C. for 24 h. The temperature was then increased to 37° C., triggering achange from aligned to unaligned fibers in the active group, while thecontrols remained unchanged. Cells were then cultured at 37° C. for anadditional 24 h.

Cell body alignment was assayed by fluorescence imaging before and afterthermal triggering. Cells were labeled with Phalloidin to visualizefilamentous actin. Two-dimensional fast Fourier transform (2D FFT) imageanalysis was used to characterize cell body alignment. The degree ofcell body alignment was determined by the amplitude of peaks in each FFTplot, with higher peaks corresponding to higher alignment in a principledirection.

Viable cells remained attached on both active and static scaffoldsbefore and after thermal triggering (data not shown). Shape-memoryactivated change in fiber alignment of the active scaffold altered cellbody alignment, with no comparable change observed in the staticcontrols. Before transition, cells cultured on the aligned fibers of theactive scaffold demonstrated two distinct peaks in the FFT plot at 90°and 270°. After transition, cells lost their preferential alignment,with no prominent peaks in the plot and corresponding reorganization ofcytoskeletal filaments was observed. In contrast, on the staticunaligned and aligned controls cells remained randomly oriented oraligned, respectively, both before and after temperature change.

Example 10—SMP Sleeve

An SMP for use as the second component of the present invention maycomprise a thermoplastic polyurethane (TPU) previously developed for useas a biodegradable stent coating. The polyhedral oligomericsilsequioxane (POSS) TPU features alternating hard segments of POSS andbiodegradable, amorphous soft segments of polylactide/caprolactonecopolymer (P(DLLA-co-CL)). To meet the specific requirements of thepresent invention, the material chemical composition was modified asfollows. A polymeric diol chain was polymerized solely from lactide (LA)monomer, instead of synthesizing from the mixture of LA monomer andanother biocompatible ester, ε-caprolactone (CL). CL was eliminated fromthe chemistry because a TPU synthesized from a LA/CL-based copolymericdiol has an undesirably low glass transition temperature (Tg) that leadsto shape recovery at a temperature substantially lower than normal bodytemperature. The synthesis was carried out in a two-step process (.Briefly, a polymeric diol of poly-DL-lactic acid (PDLLA) (hereaftercalled polyol) was synthesized by ring-opening polymerization of cycliclactide monomer (3,6-dimethyl-1,4-dioxane-2,5-dione) in the presence ofa prescribed concentration of initiating 1,4-butandiol and a smallamount of organometallic catalyst. The polyol was then reacted withhexamethylene diisocyanate (HDI) and POSS diol (AL0130, Hybrid Plastics,Hattiesburg, Miss.), also in the presence of a small amount oforganometallic catalyst. The targeted final molar ratio of polyol andPOSS diol was kept at 1:3. The glass transition temperatures of theresultant TPU synthesis batches were in the range of ˜48-49° C. Allchemicals were purchased from Sigma-Aldrich (St. Louis, Mo.) unlessotherwise noted.

To prepare structures that would allow changes of bulk shape as well asinternal fibrous architecture in response to shape-memory actuation,scaffolds were fabricated using electrospinning. The detailedelectrospinning apparatus setup was described elsewhere. Thecustom-synthesized TPU was dissolved in dimethylformamide (DMF;Sigma-Aldrich) and chloroform (Fisher Scientific, Pittsburgh, Pa.) atDMF:chloroform=1:2 (v/v). To ensure that the resultant electrospunfibers had similar diameters, the concentrations of polymeric solutionswere adjusted between different TPU synthesis batches to accommodatevariation in molecular weight, with the range of polymeric solutionconcentrations varied from 35%-45% (w/v). The polymeric solution wasloaded in a syringe with a 22 G stainless steel blunt needle used as aspinneret. The polymeric solution was pumped at a rate of 0.4 ml/hthrough the spinneret, which was positively charged to 15.5 kV. Therotating drum was negatively charged to 0.5 kV with a 10 cm distancefrom the spinneret to rotating drum surface. The total duration of theelectrospinning process was 12 h, yielding an electrospun scaffold of100 μm thickness.

To produce static control scaffolds that do not change shape or fibrousarchitecture when heated to body temperature, scaffolds of eitherrandomly oriented fibers or aligned fibers were prepared. To produce arandomly oriented scaffold, the rotation speed of the collecting drumwas set at 400 rpm. The slowly rotating drum had a negligible effect onfiber orientation in these static unaligned control scaffolds. Toproduce an aligned scaffold, the rotation speed of the collecting drumwas increased to 4000 rpm. The high-speed rotating drum preferentiallyoriented fibers along its circumferential direction in these staticaligned control scaffolds.

The electrospinning process is known to stretch amorphous polymerchains. If the electrospun scaffold is subsequently heated to near orabove the polymer glass transition temperature, the extended amorphouschains relax and release molecular-level strain, which subsequentlycauses dimensional changes in the electrospun scaffold. In the presentinvention, this premature shrinkage posed the risk of obscuring thesubsequent programmed and desired shape memory effect. To removemolecular-level strain and to prevent premature shrinkage, a novelthermal treatment was used in which, all scaffold groups were immersedin a ˜55-60 wt % Pluronic F127 thermoreversible hydrogel aqueoussolution and heated and held isothermally at 70° C. (at whichtemperature the Pluronic solution is a gel) for 3 h to release themolecular-level strain while the hydrogel stabilized the fibrousarchitectures and prevented change in scaffold shape or fiber alignment.After thermal treatment, the scaffolds were rigorously washed indeionized water at 4° C. (at which temperature the Pluronic solution isa liquid) to remove the hydrogel and were then dried in a vacuum oven atroom temperature.

To program the experimental active scaffold that can be triggered tochange shape on command, a thermally treated randomly oriented scaffoldwas uniaxially stretched to 100% strain (during which the scaffolddemonstrated contraction in the orthogonal plane with a Poisson's ratioof approximately 0.43) at 60° C. in a dynamic mechanical analyzer (DMA;TA Instruments Q800) and fixed in that temporary shape by cooling belowTg to 0° C. Active strain-aligned scaffolds prepared in this manner canbe triggered to recover from their temporarily elongated shape withaligned fiber orientation to a more compact shape with random fiberorientation by heating to 37° C. under a hydrated condition.

Scaffolds were analyzed to characterize shape memory functionality,fiber architecture and size, and macroscopic shape change undersimulated cell culture conditions. Shape memory functionality wasanalyzed using a one-way shape-memory cycle. Fiber architecture and sizewere analyzed by scanning electron microscopy (SEM) and two-dimensionalfast Fourier transform (2D FFT) image analysis (n=3 micrographs from onesample of each group; all from TPU synthesis batch 2. Macroscopic shapechange under simulated cell culture conditions was measured usingdigital calipers.

Human adipose-derived stem cells (hASCs) were used to investigate theeffect of scaffold shape and architecture change on cell attachment,viability, and morphology. This cell type was selected because ASCs andother adult (or tissue) stem cells have previously been shown todemonstrate architecture-responsive behavior on porous 3D scaffolds,including electrospun scaffolds, and because ASCs are widely used intissue engineering and regenerative medicine research and, therefore,would be likely candidates for use in future strategies employing SMPscaffolds. Prior to active cell culture experiments (experiments inwhich the scaffold was triggered to change shape and fiber architectureduring culture), hASCs (Cat# R7788-115) were expanded in complete growthmedium: MesenPro RS basal medium with 2% MesenPro RS growth supplement,1% GlutaMAX, and 1% penicillin/streptomycin in a 37° C. humidifiedincubator with 5% CO₂. Cells were cultured on a T175 flask with 30 ml ofcomplete growth medium. The medium was changed every four days and cellswere passaged at 80% confluence using TrypLE Express solution. Cellswere used at passage 6. All cell culture products were purchased fromGibco, Life Technologies (Grand Island, N.Y.).

Here, active cell culture is a two-stage process. During the firststage, cells are seeded and cultured on the active or static scaffoldsat a cytocompatible temperature (30° C.) below body temperature, atwhich temperature the active scaffolds maintain their fiber alignmentwith little change in the macroscopic shape (length) of the scaffold.During the second stage, shape memory actuation is triggered by heatingto body temperature (37° C.), resulting in changes in scaffold shape andarchitecture in the active scaffolds, while static control scaffoldsremain unchanged. To determine whether changes in scaffold shape andarchitecture would affect cell behavior, hASC cytoskeletal and nuclearalignment were characterized before and after temperature transitionduring active cell culture experiments employing active and staticscaffolds.

Active strain-aligned, static unaligned control, and static alignedcontrol scaffolds were sterilized in a biological safety cabinet underUV light for 1 h each side and then hydrated in complete growth mediumat room temperature for 20 min before seeding cells on the scaffolds.Scaffolds were then placed in 24-well plates and a droplet of cellsuspension was laid on each scaffold with a cell density correspondingto 8,000 cells/cm² for the total scaffold area. Droplet coverage, whichwas highly reproducible between samples, was marginally less than thetotal scaffold area, producing a final cell seeding density greater than8,000 cells/cm² (all downstream imaging analyses were performed onrepresentative areas within the seeded region). The plates wereincubated in a 30° C. incubator for 5 h to allow cell to attach on thescaffolds, and then an extra 500 μl of complete growth medium was addedinto each well. Two time points were selected to assay cell viabilityand morphology: during the first stage of active cell culture, cellswere assayed after being cultured at 30° C. for 24 h; during the secondstage of active cell culture, which was triggered by moving scaffolds toa 37° C. incubator, cells were assayed after being cultured for anadditional 24 h. Scaffolds were fabricated from four TPU synthesisbatches. Four scaffolds from each of the four synthesis batches wereused for the three groups—active strain-aligned, static unalignedcontrol, and static aligned control—providing a sample size of 16 (n=16)for each group at each time point.

To characterize cell cytoskeletal alignment and cell nuclear alignment,cytoskeletal actin filaments and cell nuclei were visualized by stainingwith Alexa Fluor 647 Phalloidin and SYTOX Green Nucleic Acid (MolecularProbes, Life Technologies), respectively. Samples were fixed,permeabilized, and stained following the manufacturer's instructions forphalloidin staining. Phalloidin was used at the concentration suggestedin the instructions and mixed with a 1:6000 dilution of SYTOX greenstain in phosphate buffered saline. Samples were mounted in ProLong Goldreagent for fluorescence imaging.

Cell imaging was performed using two forms of fluorescence microscopyfor distinct purposes. To quantify cell cytoskeleton and cell nuclearalignment, LIVE/DEAD and phalloidin/SYTOX green stained cells wereimaged on a Leica DMI 4000B inverted microscope outfitted with a LeicaDFC 340FX camera and using a 10×/0.22 NA objective. To acquire higherresolution micrographs suitable for qualitative analysis of cellmorphology and quantitative analysis of cell distribution,phalloidin/SYTOX green stained cells were imaged on a Zeiss LSM 710confocal laser-scanning microscope (CLSM) using a 40×/1.30 NA oilobjective with Zeiss immersol 518 F immersion oil. Red or greenpseudocolor was applied using ImageJ, with a lookup table applied to allmicrographs. Histogram stretching was applied to phalloidin/SYTOX greenimages in order to maximize printed image contrast.

To quantify cell cytoskeleton alignment on the active and staticscaffolds, a 2D FFT image analysis method was adopted and used [32].Images of cells stained with phalloidin were characterized to determinethe extent to which cell cytoskeleton alignment was controlled byscaffold architecture. A 1600×1200 px image was cropped to a square of1024×1024 px and then overlaid with a black square mask with aconcentric transparent circle (1024 px in diameter) to avoid edge/cornereffects. The masked image was computed using the FFT function in ImageJ.Pixel intensity along each radius (from 0° to 359° with 1° increment) inthe FFT plot was summed using the ImageJ plugin “Oval Profile.” Pixelintensities of the 16 images were summed. The pixel intensity of eachradius was normalized by the minimum intensity value. Subsequently, thebaseline was shifted to 0 by subtracting 1 from each value. The samplesize for analysis of cell cytoskeleton alignment was 16 (n=16). Pixelintensity was plotted with a range from 0 to 0.15.

To allow comparison of cell nuclear alignment on the active and staticscaffolds, a previously reported method was adapted to quantify thedegree of nuclear alignment. Briefly, the nuclear angle of each cell wasmeasured using ImageJ, and the standard deviation of nuclear angledistribution was determined. The standard deviation was used to quantifythe degree of cell nuclear alignment. Randomly oriented nuclear angleswould produce a standard deviation of 52°, while perfectly alignednuclear angles would produce a standard deviation of 0°. Because astandard deviation of 0° is statistically improbable for a cellularsystem of this nature, in the present work the standard deviation ofnuclear angle distribution observed on the unaligned control andrecovered-to-random samples and on the aligned control andstrain-aligned samples were used to define the effective range ofstandard deviations for unaligned and aligned cells, respectively, inthis system. The sample size for statistical analysis of cell nuclearalignment was 16 (n=16).

Cell viability was qualitatively determined using LIVE/DEAD reagents(Molecular Probes, Life Technologies). LIVE/DEAD was used at aconcentration of 2 μM for both Calcein AM and Ethidium homodimer-1.Samples were stained following the manufacturer's instructions with a 30min incubation at room temperature. The sample size for analysis of cellviability was 16 (n=16). Because cell viability in these samples wasqualitatively observed to be high in all groups 24 h before and 24 hafter temperature transition, a subsequent quantitative analysis of cellviability out to 72 h following change in scaffold shape andarchitecture was performed; samples from synthesis batch 4; n=2-3samples per group). Cell distribution within the scaffold both beforeand after shape memory actuation was determined by confocal fluorescencemicroscopy of one set of representative samples from each of thestrain-aligned and recovered-to-random groups in which cells werestained by phalloidin and SYTOX green.

The 95% confidence interval of the standard deviation of cell nuclearangle distribution was calculated using a bootstrap method.Permutation-based one factor ANOVA was performed to test for significantdifferences between the standard deviation of cell nuclear angledistributions among the three groups (strain-aligned, unaligned control,and aligned control) followed by multiple comparisons. Statisticalsignificance was determined at p<0.05. The sample size for statisticalanalysis of cell nuclear alignment was 16 (n=16).

The electrospun scaffold demonstrated desirable shape memoryfunctionality, in terms of shape fixing and shape recovery. A one-wayshape-memory cycle showed that the scaffold had a high shape fixingratio of 99%, meaning that the programmed scaffold can retain 99% of thedeformation strain after the applied load is removed. The cycle alsoshowed that the scaffold had a high shape recovery ratio of 95%, meaningthat the scaffold recovered 95% of the deformation after shape-memoryactuation.

Scanning electron microscopy revealed changes in the architecture of theshape-changing strain-aligned scaffold following thermal triggering.After the programmed uniaxial stretching, the strain-aligned scaffoldshowed prevailing fiber alignment. After incubation at 30° C. for 24 hand before shape memory actuation, the scaffold retained its temporarilyaligned fibrous architecture and showed only modest change inmacroscopic shape. After shape memory actuation by heating to 37° C.,the scaffold recovered back to a randomly oriented architecture within24 h and returned to within 20% of its original macroscopic length.

Strain-aligned and aligned control scaffolds both demonstrated aprevailing fiber alignment, as demonstrated by two distinct peaks at 90°and 270° in the FFT plots. Conversely, recovered-to-random and unalignedcontrol scaffolds had no apparent fiber alignment, as demonstrated by alack of distinct peaks in the FFT plots. In contrast to the differencein fiber alignment observed before and after shape memory actuation, thefiber diameter of strain-aligned and recovered-to-random scaffolds wasnot significantly different (p=0.29, by permutation testing).

A triggered change in scaffold architecture had a significant effect oncell cytoskeleton orientation. A change in scaffold fiber alignment viashape memory actuation was found to cause cells to change frompreferential alignment of actin filaments along the fiber direction to amore random orientation. Before triggering scaffold architecture change,two distinct peaks at 90° and 270° in the FFT plot indicated that actinfilaments were aligned along a principle direction, which was thescaffold fiber direction. After transition, actin filaments lost theirpreferential alignment, as indicated by a lack of distinct peaks in theFFT plot. Control groups that did not change scaffold architectureconfirmed that cell cytoskeleton reorientation was induced by a changein scaffold fiber alignment rather than by the temperature transition.The FFT plots showed a lack of distinct peaks for the unaligned controland two distinct peaks for the aligned control, both before and aftertemperature transition. Although FFT peak height is not an accurateindicator of alignment—with width at half height being instead a moreaccurate measure of alignment—the shape of the FFT plot for the alignedcontrols before and after temperature change does suggest that thecytoskeletal organization changed subtly between the first time point(at 30° C.) and the second time point 24 h later (at 37° C.). Whetherthe subtle changes in cytoskeletal organization on these controlscaffolds is due to changes in cell behavior over time, to the change intemperature, or to a combination of the two remains to be determined.Regardless, the controls confirmed that cell cytoskeleton reorientationon the experimental, architecture-changing groups was induced by achange in scaffold fiber alignment rather than simply by the temperaturetransition.

A triggered change in scaffold architecture also had a significanteffect on cell nuclear alignment. Angular histograms showed a narrowdistribution of cell nuclear angles on active strain-aligned and alignedcontrol scaffolds and a broad distribution of cell nuclear angles onrecovered-to-random and unaligned control scaffolds. Before triggeringof scaffold architecture change, cell nuclei preferentially aligned inthe direction of scaffold fiber direction with a standard deviation ofcell nuclear angle distribution of 41.87±3.21°. After triggering achange in scaffold fiber alignment, cell nuclei became more randomlyoriented with a standard deviation of cell nuclear angle distribution of47.43±1.71° (p=0.001). Cell nuclear alignment remained unchanged for thetwo static control groups. Before and after shape memory actuation, thestandard deviation of nuclear angle distribution for unaligned)(˜46-47°) and aligned) (˜35-37°) control scaffolds respectively definedthe effective range of standard deviations for unaligned and alignedcells in this system.

Qualitatively, LIVE/DEAD assay showed cells were viable in all groupsbefore and after temperature transition. Cells that were seeded on theactive strain-aligned scaffold remained viable and attached aftershape-memory-actuated scaffold fiber alignment change. Quantitativeanalysis of cell viability out to 72 h following change in scaffoldshape and architecture showed that cell viability remained greater than86% for all groups at all time points. Although this study was notdesigned to reveal the limit of tolerable deformation, or, in fact,ranges of cellular responses to different deformation levels, cells werefound to remain attached and viable even when the strain-alignedscaffold was fixed at 200% strain (rather than the 100% strain usedthroughout this study). Confocal fluorescence microscopy showed thatcells infiltrated 21 μm or more (two to three cell depths) into thescaffold during the first 24 h of culture at 30° C., at which pointshape-memory recovery was triggered. After culture for an additional 24h at 37° C. during and following scaffold shape-change, cells hadinfiltrated 42 μm or more (four to six cell depths) into the 100 μmthick scaffold.

Example 11—SMP Sleeve

Preliminary in vivo results in a mouse femoral segmental defect modelhave demonstrated that the TPU sleeve of the present invention may tearat the bone-graft junctions. One approach to reinforcing the TPU sleeveis to incorporate a semi-crystalline polyester, poly(L-lactic acid) orPLLA, during sleeve fabrication via a co-electrospinning process. PLLAfibers may be blended with TPU fibers at 20% volume ratio (controlled byvarying solution feeding rate) throughout the entire thickness of thesleeve. The stability provided by the PLLA-TPU sleeve will be assessedby torsional and four-point bending mechanical tests. Another approachis to add a mesh of suture to the center of the tube to provideadditional mechanical rigidity and tear resistance. Sutures are laidacross the material at a specified pitch in both a clockwise andcounterclockwise direction. This reinforcement will provide increasedmechanical rigidity and tear strength, while not reducing the beneficialporosity of the material.

Example 12—Cell Culture Substrate

In this example, a co-polymer system with tert-butyl acrylate (tBA) andbutyl acrylate (BA) was used for producing substrates capable ofprogrammed topography transition suitable for controlling cellmorphology with the intent of lineage specification for periosteogeniccell culture and tissue engineering Specifically, a buckling phenomenaupon contraction of a thin, rigid material atop a thick, compliant layerwas used to produce nanoscale wrinkles whose dimensional propertiesdepend on the amount of contraction and thickness and stiffness of eachlayer. The film was cured with a transition temperature near 45° C. dry,which lowered when plasticized by water. This 1 mm thick film was thencut to 6 mm×24 mm and stretched to 12% uniaxial tensile strain in a DMA.This strain was fixed by holding the strain and cooling to 10° C. Theprogrammed substrate was then placed into a gold sputter coater andsputter coated for 100 s to produce a thin, rigid layer atop the SMPfilm. Human adipose derived stem cells were plated on this substrate andcultured at 30° C. for 5 h. Topography change was found to control stemcell morphology, as intended. Cell nuclear and filamentous actinstaining of human adipose derived stem cells plated on the temporarilyflat substrate was randomly oriented. Following move of the substrate toa 37° C. incubator and cultured for 24 h, which results in thetopographic trigger to the wrinkled topographic surface, the stem cellsbecame preferentially oriented.

Example 13—Cell Culture Substrate

Using a co-polymer system with tert-butyl acrylate (tBA) and butylacrylate (BA), the effect of cell seeding density on both the expandingsubstrates and topography changing (wrinkling) SMP substrates wasstudied. Both multipotent C3H10T1/2 mouse embryonic fibroblasts andmultipotent human adipose derived stem cells (hASCs) were studied, asthese two multipotent cell types both have the potential for use in anin vivo mouse model. Briefly, cells were seeded at either 10⁵ or 2×10⁵cells per ml on the gradient expanding or topography changing(wrinkling) substrates and cultured at 30° C. to confluence, at whichtime the substrates were triggered to change shape by warming to bodytemperature (37° C.). Cell morphology and behavior were analyzedqualitatively by light microscopy at 1, 3, 4, 5, 6, 7, and 8 days ofculture.

Both cell types attached to and proliferated on the expanding andtopography changing (wrinkling) substrates. hASCs showed apparent weakerattachment to the substrate, with cells tending to form nodules and cellsheets delaminating over time, and reduced proliferation compared to theC3H10T1/2 cells.

To determine whether improved cell attachment would improve hASCproliferation and spreading, hASCs were subsequently seeded on gelatincoated samples at densities of 2×10⁵ or 3.3×10⁵ cells per ml. Theimproved cell attachment provided by the gelatin coating was found toresult in more uniform cell attachment, spreading, and proliferation,indicating that the substrates are suitable for use with hASCs, givenappropriate surface modification to enable robust attachment.

Cell viability and adhesion on bare (unmodified) substrates were foundto be suitable and gelatin coating significantly improved the attachmentof hASCs. hASCs are preferred as the cell type to be employed in the invivo critically sized defect mouse model, with substantial literaturesupporting the implantation of hASCs in immune competent mice.

Cell cultures prepared according to the present invention may be gaugedusing measurable metrics, such as Alk Phosphatase, cellular alignmentand morphology, cellular density, and/or mineralization, to determinetheir appropriateness for incorporation into the system. For example,cell cultures that would be appropriate for use in the present inventionexhibit increased staining for alkaline phosphatase, increased cellularalignment, increased nuclear alignment as well as cellular and nuclearaspect ratios, increased cell density; and increased mineralized noduleformation (which can be assayed by Xylinol Orange staining).

Example 14—Cell Culture Substrate

An automated tracking algorithm was designed to process an image stackof stained nuclei and accurately identify, track, and analyze cellmotility over long timescales. In this algorithm, cell nuclei are firstsegmented using a contour-based approach that incorporated cell mergingand division capabilities. Interacting cells (cells that merge ordivide) are established by isolating contour profiles with multiple-peakintensities and identifying dual peaks (two cells) that share a parentcontour. Following tagging of interacting cells, linking is achievedthrough the use of a particle tracking approach. After tracking iscomplete, detailed merging and division events are constructed throughanalysis of cell interactions and cell tracks are corrected using acustomized cost function. Finally, the high-accuracy tracking enabled bythe correction of merging and division events is leveraged inquantitative analyses, in which the updated tracks are sent through aseries of correlation functions to quantify cell motility behavior,including mean squared displacement (MSD) and velocity autocorrelation.

The tracking code was applied to the study and analysis of cell motilityon the complex topography changing wrinkle substrates, which have a highorder of anisotropy. Gold-coated substrates with micron-scaled wrinkleswere used to investigate the effect of a highly anisotropic topographyon cell motility behavior, and those results were compared to cellmotility behavior atop gold-coated substrates with a flat topography.Tissue culture polystyrene (TCPS) was used as a control substrate toensure that the material was not indicative of specific behaviors.Images with poor contrast were acquired, which experimentally enabledcell divisions to occur and analytically tested the robustness of thetracking code. Experiments at three different cell densities wereconducted to test the segmentation and merging/division processing ofthe code as confluence is approached.

Cell culture experiments were conducted by seeding C3H10T1/2 mousefibroblasts on wrinkled, non-wrinkled, and TCPS substrates. Cells wereseeded using a droplet method. Cell solutions with concentrations of87,500, 175,000, and 262,500 cells per ml were prepared. These densitiescorrespond to increasing cell-to-cell interactions, further testing thecapabilities of the developed approach and its ability to process moredense environments. In each case, a 20 μL droplet of cell solution wasseeded onto the substrates and samples were then placed in a 37° C.incubator for 2 h to allow for cell attachment. After 2 h, completegrowth medium was added and the samples were placed in a 37° C.incubator for an additional 22 h, after which point the cells werestained and prepared for live cell imaging.

Cell tracks were plotted to visualize collective cell motility behavior.Qualitatively, wrinkled trajectories displayed directionalitycorresponding to the wrinkled direction, while non-wrinkled topographiesdisplayed random trajectories.

Mean squared displacement and velocity profiles were characterized. Meansquared displacement values showed similar motility behavior at shorttimescales. However, at long time scales, the lowest density exhibiteddistinct differences in motility behavior. For all wrinkled velocityprofiles, a clear separation between x and y correlations indicatedpreferential migration along the wrinkled direction. For non-wrinkledvelocities, this directional motion was not apparent.

The topography changing substrates successfully controlled stem cellmorphology. The intent is for this morphological control to be employedto control cell differentiation and cell sheet engineering, such as bytaking advantage of the osteogenic potential of two differenttopographic transitions, namely, the flat to nano-scale wrinkledtransition and a transition from a flat topography to a square-wavetopography of parallel plateaus 15 μm wide spaced 15 μm apart and 5 μmhigh.

Example 15—In Vivo Assessment

A mouse critically sized defect model may be used for in vivoassessment. For example, a graft may be implanted into a 4 mm defectcreated in the middiaphysis of the femur of a recipient mouse andsecured with an intramedullary pin. Three experimental groups may beexamined: the scaffold of the present invention alone, the scaffold incombination with the sleeve of the present invention, and the scaffoldin combination with the sleeve and the periosteal sheets of the presentinvention.

What is claimed is:
 1. A system for repairing a bone defect, comprising:a porous foam scaffold comprised of a shape memory polymer selected fromthe group consisting of poly(tert-butyl acrylate/butyl acrylate),poly-DL-lactide, and poly-DL-lactide/polyhedral oligomericsilsesquioxane that is configured for expansion in response to a firststimulus after being positioned within a bone defect; a sleeve comprisedof a second shape memory polymer having reinforcing fibers therein thatis configured for radial contraction in response to a second stimuluspositioned around said bone defect and said porous foam; and a sheet ofcells cultured on a shape memory polymer substrate to have a morphologyconducive to vascularization applied to said sleeve.
 2. A system forrepairing a bone defect, comprising a porous foam scaffold comprised ofa shape memory polymer that is configured for expansion in response to afirst stimulus after being positioned within the bone defect and havinga pore size of 200 to 500 micrometers and a porosity of 79 percent plusor minus five percent.
 3. The system of claim 2, wherein said porousfoam comprises a polymer selected from the group consisting ofpoly(tert-butyl acrylate/butyl acrylate), poly-DL-lactide, andpoly-DL-lactide/polyhedral oligomeric silsesquioxane.
 4. The system ofclaim 2, further comprising a sleeve comprised of a second shape memorypolymer that is configured for radial contraction in response to asecond stimulus positioned around said bone defect and said porous foam.5. The system of claim 4, wherein said sleeve comprises a thermoplasticpolyurethane.
 6. The system of claim 5, wherein said sleeve includessemi-crystalline polyester fibers.
 7. The system of claim 6, wherein thesemi-crystalline polyester fibers comprise poly(L-lactic acid).
 8. Thesystem of claim 2, further comprising: a sleeve comprised of a secondshape memory polymer that is configured for radial contraction inresponse to a second stimulus positioned around said bone defect andsaid porous foam; and a sheet of cells having a morphology conducive tovascularization applied to said sleeve.